Abstract

Memory type 1 T helper (T(H)1) cells are characterized by the stable expression of interferon (IFN)-γ as well as by the epigenetic imprinting of the IFNG locus. Among innate cells, NK cells play a crucial role in the defense against cytomegalovirus (CMV) and represent the main source of IFN-γ. Recently, it was shown that memory-like features can be observed in NK cell subsets after CMV infection. However, the molecular mechanisms underlying NK cell adaptive properties have not been completely defined. In the present study, we demonstrated that only NKG2Chi NK cells expanded in human CMV (HCMV) seropositive individuals underwent epigenetic remodeling of the IFNG conserved non-coding sequence (CNS) 1, similar to memory CD8(+) T cells or T(H)1 cells. The accessibility of the CNS1 was required to enhance IFN-γ transcriptional activity in response to NKG2C and 2B4 engagement, which led to consistent IFN-γ production in NKG2C(hi) NK cells. Thus, our data identify epigenetic imprinting of the IFNG locus as selective hallmark and crucial mechanism driving strong and stable IFN-γ expression in HCMV-specific NK cell expansions, providing a molecular basis for the regulation of adaptive features in innate cells.

(A) Alignment of the human IFNG and mouse Ifng locus as presented by VISTA browser, DNA sequence identity >50% over at least 100 bp. Conserved regions with >70% sequence identity are marked in red and are depicted relative to the IFNG/Ifng transcriptional start site (TSS). Arrow indicates transcription direction and exon/UTR regions are indicated in blue. (B–D) Methylation status of the IFNG promoter and/or CNS1 was analyzed by determining CpG methylation of isolated DNA by bisulfite pyrosequencing. Five CpGs from −53 to +171 bp located in the IFNG promoter and six CpGs from −4399 to −4278 bp in the CNS1 region were analyzed and mean percentage of methylation at each individual CpG is depicted. (B and C) CpG methylation of naïve CD4+ T cells, TH1 cells and NK cells, FACS sorted ex vivo as described in . One representative experiment out of two (T cells) or out of three (NK cells) is shown. (D) FACS sorted total NK cells were labeled with 500 nM CFSE and cultured in the presence of the indicated cytokines. After five days, viable CFSElo NK cells, which have undergone proliferation, were FACS sorted and the methylation status of the IFNG CNS1 was analyzed. Mean percentage of methylation ± SEM at each individual CpG is depicted (n = 3).

Analysis of surface marker expression and methylation status of the IFNG promoter and CNS1 was analyzed as described in in ex vivo NK cell subsets derived from representative HCMV− (n = 2) (A) or HCMV+ (n = 4) donors (B and C), without (B) or with expansion of NKG2Chi NK cells (C). NKG2C and CD57 expression was analyzed by FC on PBMC, after gating on viable CD3− CD56dim NK cells, while sKIR (KIR2DL3 in HLA-C1+ donor) expression was analyzed after gating on viable CD3− CD56dim CD57+ NKG2C+/− NK cells. CpG methylation of the IFNG promoter and CNS1 was analyzed in FACS sorted CD56dim CD57+ NKG2C− (gray bars) and in CD56dim CD57+ NKG2C+/hi NK cells (black bars) from each donor and is depicted as mean percentage of methylation at each CpG site.

(A–C) Phenotype and methylation status of the CNS1 was analyzed in ex vivo NKG2Chi NK cell expansions from representative HCMV+ donors (n = 4), as described in . (A and B) CD56 and NKG2C expression was analyzed by FC on PBMC, after gating on viable CD3− CD56+ NK cells, while CD57 and sKIR (KIR2DL3 in HLA-C1+ donor) expression was analyzed after gating on CD56dim NKG2Chi NK cells. CpG methylation of the IFNG CNS1 was analyzed in FACS sorted CD56dim NKG2C+ NK cell subsets as indicated and is depicted as mean percentage of methylation at each CpG site. (C) The same HCMV+ individual displaying an expanded NKG2Chi population was analyzed twice with an interval of one year between the two measurements. NKG2C and CD57 expression was analyzed by FC on PBMC, after gating on viable CD3− CD56dim NK cells, while sKIR expression was analyzed after gating on CD56dim CD57+ NKG2C+/− NK cells. CpG methylation of the IFNG CNS1 was analyzed in FACS sorted CD56dim CD57+ NKG2C− (gray bars) and in CD56dim CD57+ NKG2C+ NK cells (black bars) and is depicted as mean percentage of methylation at each CpG site.

Genomic DNA of ex vivo FACS sorted NK and T cell subsets was analyzed for CpG methylation by RRBS. (A–C) Beta-values for each methylation site identified by RRBS were averaged from two donors for each T or NK cell subset as indicated. Sites with coverages below 5 were removed from further analysis. (A) Clustering analysis of differentially methylated CpG sites found by RRBS. Clustering was performed on 1,000 most variant methylation sites out of 30,000 randomly chosen sites, localized within gene bodies and promoter regions. The indicated cell subsets cluster according to the similarities of their methylation profile. Beta-values depicted from dark-red to yellow represent level of methylation of the individual CpG sites. (B) Similarities between the cell subsets as judged by the Euclidian distance. (C) Principle component analysis (PCA) of mean methylation levels from two donors for indicated cell subsets. The PCA revealed a clear separation of CD4+ TH1 and CD8+ memory cells, naïve CD4+ and CD8+ cells, and NK cells by the first two components. (D) CpG methylation sites of T and NK cell subsets of one representative donor identified by RRBS in gene bodies of TXB21, EOMES, PRF1, TNF, PRDM1 and ZBTB32. Line diagram represents sequence conservation within vertebrates. Blue and violet bars indicate the methylation level (0–100%) and coverage for each identification, respectively. Coverages of 5 and more reads are represented as a full violet bar.

(A) Surface expression of NKG2C and 2B4 on NKL (black line) or isotype control (solid grey histogram) was determined by FC. (B) Intracellular expression of IFN-γ by NKL was detected by FC after crosslinking of NKG2C and/or 2B4 for 16 hours. One representative experiment out of four is depicted. (C and D) Luciferase reporter assay of IFNG sequences transfected in NKL. (C) Construct containing the IFNG promoter (IFNGp) region was cloned into the Luciferase reporter vector pGL3 upstream of the Firefly luciferase gene (Luc). pGL3 reporter vectors were transfected into NKL cells along with Renilla reporter vector pRL-TK as internal control and luciferase activity was measured after stimulation of NKL, as indicated. Relative luciferase units (RLU) were calculated in relation to the activity of the IFNGp (−571 to +71) stimulated with aNKG2C+a2B4, after normalization to Renilla luciferase and basic pGL3 activity. Mean RLU ± SEM (n = 3) are depicted. (D) Constructs containing the IFNGp (−49 to +71) region with or without the CNS1 were cloned into the CpG-free vector pCpGL. Luciferase activity of untreated (unmethylated, open circles) and of CpG-methyltransferase (M.SssI)-treated vectors (methylated, black circles) was measured after transfection into NKL cells. RLU were calculated relative to the activity displayed by the unmethylated CNS1+IFNGp (−49 to +71) vector stimulated with aNKG2C+a2B4, after normalization to Renilla luciferase and basic pCpGL activity. One representative experiment out of three is depicted.